A striking feature of Cant Syndrome(CS) is that over 50% of patients present with some form of lymphedema. Although the underlying disease process in CS is known to involve gain-of-function (GOF) mutations in the KATP channel, the mechanism by which lymphedema develops in CS patients is unknown and has not been studied previously. Because spontaneous contractions of collecting lymphatic vessels underlie ? of normal lymph transport, we propose that lymphedema in CS, and potentially other forms of lymphedema, result from reduced excitability of lymphatic smooth muscle. Our central hypothesis is that CS-related GOF mutations in Kir6 and/or SUR subunits result in hyperactivation of KATP current, hyperpolarization of lymphatic smooth muscle and inhibition of the intrinsic electrical pacemaker that drives spontaneous lymphatic contractions. We have pioneered methods to test our hypothesis using popliteal lymphatic vessels of the mouse. Diameter and indices of pumping efficiency are measured in single, cannulated vessels under defined pressure / flow conditions ex vivo. We record membrane potential in either lymphatic smooth muscle or endothelium using sharp electrodes, and dissociate and patch clamp either cell type in order to selectively assess KATP current. In complementary in vivo studies using conventional or near-infrared fluorescence imaging, we assess the function of multi-valve chains of popliteal vessels so that potentially beneficial effects of classic and novel KATP channel antagonists can be tested. Our ability to study mouse lymphatic vessels allows us to take advantage of the global and tissue- specific KATP channel gain- or loss-of-function mutants generated by our collaborators. We have collected a substantial amount of preliminary data to show that Kir6.1 and SUR2 isoforms of KATP are functionally expressed in mouse lymphatic vessels and that spontaneous lymphatic contractions in Kir6.1 or SUR2 GOF mice are severely attenuated but can be rescued by inhibition of KATP channels. These preliminary data are the first demonstration of an underlying contractile defect leading to primary lymphedema. The central hypothesis will be tested with 3 aims. 1) Delineate how KATP channels normally regulate lymphatic pumping. We will assess the roles of LSM and/or LEC KATP channels in mediating inhibition of pumping by nitric oxide and prostanoids. 2) Determine how CS GOF KATP mutations impair lymphatic pumping by expressing the mutant channels in the LEC / LSM layers and measuring excitability and contractile function. 3) Assess whether lymphatic dysfunction can be rescued in mice with CS GOF KATP mutations. The identified source of lymphatic dysfunction in CS (a possible contractility deficit) provides a novel opportunity to test if pharmacologic intervention can be effective in reversing CS-related lymph transport dysfunction. Accomplishment of these aims will elucidate the role of KATP in lymphatic vessels, uncover the mechanism of lymphatic dysfunction in CS, and test a novel therapeutic method to treat lymphedema in CS and other patients with reduced lymphatic smooth muscle excitability.